METHOD FOR PRODUCING PROTEIN INCLUDING k CHAIN VARIABLE REGION

Abstract

A method for producing a protein includes adsorbing a protein including a κ chain variable region on an insoluble carrier of an affinity separation matrix by contacting a liquid sample including the protein with the affinity separation matrix; washing the affinity separation matrix to remove impurities; separating the protein from the affinity separation matrix by using an acidic buffer; and regenerating the affinity separation matrix by using an alkaline aqueous solution after the protein is separated from the affinity separation matrix. The insoluble carrier includes a ligand immobilized on the insoluble carrier, and the ligand is a κ chain variable region-binding peptide including B5 domain of Protein L derived from Peptostreptococcus magnus 312 strain or a variant of the B5 domain. The adsorbing, the washing, and the separating are repeated 3 or more times.

What is claimed is: 
     
         1 . A method for producing a protein, comprising:
 adsorbing a protein comprising a κ chain variable region on an insoluble carrier of an affinity separation matrix by contacting a liquid sample comprising the protein with the affinity separation matrix;   washing the affinity separation matrix to remove impurities;   separating the protein from the affinity separation matrix by using an acidic buffer; and   regenerating the affinity separation matrix by using an alkaline aqueous solution after the protein is separated from the affinity separation matrix,   wherein the insoluble carrier comprises a ligand immobilized on the insoluble carrier,   wherein the ligand is a κ chain variable region-binding peptide comprising B5 domain of Protein L derived from  Peptostreptococcus magnus  312 strain or a variant of the B5 domain, and   wherein the adsorbing, the washing, and the separating are repeated 3 or more times.   
     
     
         2 . The method according to  claim 1 , wherein the regenerating is also repeated 3 or more times after the separating. 
     
     
         3 . The method according to  claim 1 ,
 wherein an amino acid sequence of the B5 domain is an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 16, and   wherein an amino acid sequence of the variant of the B5 domain is selected from the group consisting of:   an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 16 comprising a total of 1 to 10 amino acid deletions, substitutions and/or additions, wherein the amino acid sequence has a binding ability to the κ chain variable region; and   an amino acid sequence having a sequence identity of 85% or more with the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 16, wherein the amino acid sequence has a binding ability to the κ chain variable region.   
     
     
         4 . The method according to  claim 3 ,
 wherein the κ chain variable region-binding peptide comprises the variant of the B5 domain, and   wherein the amino acid sequence of the variant of the B5 domain is an amino acid sequence corresponding to SEQ ID NO: 7, wherein the 17 th  position is Glu, the 19 th  position is Ile, the 20 th  position is Tyr, the 22 nd  position is Glu, the 25 th  position is Thr, the 26 th  position is Val, the 30 th  position is Thr, the 50 th  position is Ser and the 53 rd  position is His.   
     
     
         5 . The method according to  claim 3 ,
 wherein the κ chain variable region-binding peptide comprises the variant of the B5 domain, and   wherein the amino acid sequence of the variant of the B5 domain is an amino acid sequence corresponding to SEQ ID NO: 16, wherein the 7 th  position is Glu, the 9 th  position is Ile, the 10 th  position is Tyr, the 12 th  position is Glu, the 15 th  position is Thr, the 16 th  position is Val, the 20 th  position is Thr, the 40 th  position is Ser and the 43 rd  position is His.   
     
     
         6 . The method according to  claim 3 , wherein the ligand is a multimer of the B5 domain or the variant.